The invention relates generally to methods and materials for the genetic analysis of an individual organism.
For some species, reliable, simple technologies are available for genetic analysis of individuals. However, for most animal and bird species genetic information is insufficient for applied genetics. Developing existing technologies for each species to obtain genetic data will be extremely laborious and time consuming. Progress to date has been slow. The situation is particularly problematic in the area of wildlife management. For example, building DNA-patterns of hawks is currently almost impossible. At the same time, there have been reports of people illegally placing eggs from wild mating hawk couples in tamed breeding hawk nests. It is currently nearly impossible to prove fraud using DNA-data in species where genetic variation has not been previously described.
Other areas of interest are DNA-identification of exotic species (e.g., animals, plants, organisms) for various reasons. For instance, animals arriving through the veterinary control can be identified by sampling them both at departure, as well as at arrival. Using the animal""s individual DNA to identify it, animals can be tracked, and proof of their origin always possible.
Furthermore, parentage verification in rare, expensive, animals and strain identification of plants can be performed for any given combination or species. Reports have been made of selling the offspring of xe2x80x98lowerxe2x80x99 breeding parents as the highest possible quality animals.
Another problem is the determination of sex. For many exotic species, genetic markers are not available to perform sex determination.
A need exists for a method of quickly genetically analysing a species to determine, among other things, its lineage, sex, and origin.
The invention provides a new technology which has been developed for the quick genetic analysis of a species and individuals thereof. The method includes the use of first and second oligonucleotide primers for performance of a PCR amplification on the genomic DNA. The first oligonucleotide primer is a 5xe2x80x2 variation generator, including a repeat sequence and a least one non-repeat nucleotide. The second oligonucleotide primer is a 3xe2x80x2 fragment generator starting within such a genetic distance that amplification of the genomic DNA can be performed, and preferably includes inosine. A PCR amplification of the genomic DNA is conducted at a relatively low annealing temperature using both the first and second oligonucleotide primers under conditions such that essentially neither the first nor the second oligonucleotide primer alone can amplify sufficient DNA to be detected. DNA fragments are thus produced based on repeat sequences on one end of the genomic DNA, and other sequences based on the opposite end of the genomic DNA. The resulting PCR products can then be analysed for the length of a repeat sequence found in the genome. A second PCR is preferably conducted on the diluted PCR products of the first PCR. Such a second PCR would be conducted using third and fourth oligonucleotide primers. The third and fourth oligonucleotide primers are elongated versions of the first and second oligonucleotide primers, respectively, thus enabling PCR amplification at relatively higher annealing temperatures, and enabling a selection of a sub-set of the DNA fragments amplified in the first PCR. At any point, an optional but preferred restriction digestion may be conducted. The technology has been developed for the quick genetic analysis of a species which is reliable, reproducible, simple, and useful for all species/organisms (e.g., animal, avian, bacterial, viral, and plant). The invention particularly relates to samples obtainable from non-human species but is applicable to samples obtained from humans as well. Neither variation in genome length nor genome composition appears to influence or limit the characteristics of the technology. The new technology is generally reliable, reproducible, simple, and useful for all species/organisms (e.g., animal, avian, bacterial, viral, and plant). Furthermore, any material containing DNA (e.g., blood, hair follicles, etc.) can be used as a source for the generation of DNA-patterns.
In one aspect, the invention includes a method of analysing genomic DNA in a sample. This method includes providing first and second oligonucleotide primers, wherein the first oligonucleotide primer is a xe2x80x9c5xe2x80x2 variation generatorxe2x80x9d comprising a repeat sequence and at least one non-repeat nucleotide on the first oligonucleotide""s 5xe2x80x2 end. Meanwhile, the second oligonucleotide primer is a xe2x80x9c3xe2x80x2 fragment generatorxe2x80x9d starting within such a genetic distance that amplification of the genomic DNA can be performed. A nucleic acid amplification such as a polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) amplification is conducted on the genomic DNA in the sample using both the first and second oligonucleotide primers. The nucleic acid amplification is conducted under conditions such that neither the first nor the second oligonucleotide primer alone amplifies DNA, thus producing DNA fragments based on repeat sequences on one end of the genomic DNA and other sequences based on the opposite end of the genomic DNA. The amplified products are then analysed to determine the length of a repeat sequence found in said genomic DNA, which can be compared with the DNA putatively of the same individual or the DNA of the individual""s putative ancestors or relatives.
Alternatively, and as more thoroughly described hereinafter, multiple amplifications and/or restriction digestion might also be used with the technique.
As described, the first primer, the xe2x80x9c5xe2x80x2 variation generatorxe2x80x9d, includes a complementary repeat sequence and at least one non-repeat nucleotide so as to start the amplification at a repeat sequence of the genomic DNA.
By localising the 5xe2x80x2 variation generator at the 5xe2x80x2 site of repeat sequences, the repeat length variation is enclosed in the amplification rounds. Primers are thus bound to hybridise at the 5xe2x80x2 ends of repeat sequences by addition of one or more nucleotides at the end of the primer.
While the oligonucleotide primers at repeat sequences provide detection of genetic variation, the 3xe2x80x2 fragment generator is used to amplify fragments of reasonable sizes (e.g., 100 base pairs to 10 kb). The 3xe2x80x2 fragment generator starts within such a genetic distance, that amplification of a sample DNA can be perform, and preferably includes inosine or another a-selective base allowing to influence annealing temperatures without coincident or equal influence on the stringency of the annealing reaction. The 3xe2x80x2 fragment generator is designed to anneal to the DNA within a short distancexe2x80x94as mentioned before. To do this, the number of selective nucleotides is kept at a low number, whereas the annealing temperature is influenced by a number of non-selective nucleotides, such as inosines, universal bases, and any combination of A,C,G or T (e.g. R,Y,N. By providing the use of such a 3xe2x80x2 fragment generator the invention provides optimal reaction conditions in the reaction, generally well suited to the reaction conditions required for the 5xe2x80x2 generator. In short, the number of selective nucleotides of this primer is maintained at a relatively low number, whereby the annealing temperature is raised to enable reliable and reproducible amplification, using a-selective bases such as inosines in the fragment generator oligonucleotide.
Some of the genetic markers identified using the technology will be located on the male and female sex-chromosomes. After the identification of such markers, these markers can be used to determine the sex of species, which are difficult to establish through phenotypic characteristics (e.g., porcupine or crocodile).
The invention also includes a kit of parts for performing the genetic analysis, and a method of manufacturing such kit for use in genetic analysis. The invention is further described in the detailed description without limiting the invention thereto